Plasma exosome extraction: can it be done with a 200 µL sample? Looking for a low-sample-volume protocol.

I have encountered the issue of large elution volumes with imported column-based methods. Domestic membrane affinity columns are actually more flexible in this regard, and many can achieve elution in 80 ul. Contact the manufacturer to request extraction data for small-sample plasma, especially looking at the alignment rate after miRNA sequencing library preparation and exogenous spike-in recovery. I suggest using your existing samples to run 3 replicates: imported column-based method, domestic column-based method, and if necessary, domestic magnetic bead-based method. Within budget, you can discuss trial kits, with cold-chain delivery to Shanghai in 1-2 days. Be sure to ask whether the kit includes DNase treatment, otherwise gDNA will interfere with miRNA quantification.

For miRNA sequencing from 200-300 µL plasma, I would not recommend direct precipitation, as background and yield are unstable. Prioritize membrane-affinity spin columns or magnetic bead methods with a small elution volume, requiring the elution volume to be controlled at 50-100 µL to avoid subsequent concentration. Before purchasing, ask the manufacturer whether they can directly extract exosomal RNA, or extract exosomes followed by lysis and then use a microRNA column, as a one-step method saves sample. In Shanghai, SF cold chain usually takes 1-2 days, so have the supplier ship next-day delivery. With a budget of 50-100 RMB per sample, domestic options are available.

Your volume is very limited, so the preprocessing needs to be minimized. First, centrifuge the plasma at 4°C and 3000g for 10 minutes to remove platelets and cell debris; do not subject it to repeated freeze-thaw cycles. After extraction, use a Bioanalyzer or capillary electrophoresis to assess small RNA; do not focus only on the RIN, because plasma exosomal RNA sometimes has a low RIN but a usable small RNA peak. Add a known miRNA spike-in before extraction to monitor yield and inhibitors. If the elution volume is still large, do not exceed 30°C during vacuum centrifugal concentration to avoid RNA degradation.

After extracting RNA from 200 µL of plasma, the yield may not be sufficient for library construction. At least 500 µL is recommended, unless a dedicated micro-scale kit is used.

I used a certain imported serum exosome kit, and it can also extract from 150 ul, but the particle count is low, and it is barely enough for qPCR.